Fragile X mental retardation protein interactions with the microtubule associated protein 1B RNA

Abstract

Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.Keywords: FMRP; fragile X syndrome; G quadruplex; MAP1B RNA; RGG boxThe lack of fragile X mental retardation protein (FMRP) results in the fragile X syndrome, the most prevalent inherited mental disorder (Crawford et al. 2001). The absence of FMRP is due to the transcriptional inactivation of the fmr1 gene, caused by an unstable expansion of a CGG trinucleotide repeat in its 59-untranslated region (UTR) (Jin and Warren 2000;O'Donnell and Warren 2002). The function of FMRP has been extensively studied, however its cellular role and how its loss causes mental retardation is still not fully understood. It is believed that FMRP is a translational regulator for specific messenger RNAs (mRNAs), and it has been shown that it is associated with actively translating ribosomes (Antar and Bassell 2003;Jin and Warren 2003;Massimiliano et al. 2004). Nucleic acid chaperone properties have also been attributed to this protein (Gabus et al. 2004). This 632 amino acid protein has two types of RNA binding domains: two K-homology (KH) domains and one arginine-glycine-glycine (RGG) box, suggesting that FMRP exerts its function through RNA binding (Siomi et al. 1993).Several studies reported that FMRP uses its RGG box to bind with high affinity to mRNAs rich in G content that could adopt more complex G quadruplex structures Darnell et al. 2001;Schaeffer et al. 2001;Ramos et al. 2003). Formed from four guanine residues, a G quartet has a planar conformation stabilized by Hoogsteen base pairs (Fig. 1A). Several such planar structures can stack to form G quadruplexes, which are stabilized by potassium or sodium cations, but are disrupted in the presence of lithium cations (Davis 2004;Hazel et al. 2004;Mergny et al. 2005). It has also been shown that FMRP uses its KH2 domain to bind to a synthetic RNA that harbors a loop-loop kissing complex (Darnell et al. 2005). However, despite these extensive efforts, the in vivo...

Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5′-Untranslated Region

“…FMRP binds tightly to G-quadruplexes in the 5Ј-UTRs of protein phosphatase 2A catalytic subunit and MAP1B (microtubule-associated protein 1B), and it was suggested that this interaction represses translation of both mRNAs (77,80). Furthermore, it was demonstrated that the FMRP-G-quadruplex repressor in the MAP1B 5Ј-UTR was destabilized by an increased FMRP concentration, suggesting that the variation of FMRP concentration in response to neuronal stimulation might act as a regulatory switch from translational repressor to a translational activator (43). The RGG box of FMRP was shown to be responsible for the interaction with the G-quadruplex secondary structure in the MAP1B 5Ј-UTR (43).…”

“…There is growing evidence that the formation of G-quadruplex secondary structures within 5Ј-UTRs results in translational repression of a number of different mRNAs, including N-ras, Zic-1, membrane-type matrix metalloproteinase 3, ESR-1, and Bcl-2 (35)(36)(37)(38)(39)(40)(41)(42). Similar to the IRE stem loop, the stability of G-quadruplex structures could be modulated by RNA binding proteins such as FMRP or members of the hnRNP A family (43,44).…”

Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

“…Naturally occurring G-quadruplexes targeted by FMRP adopt multiple conformations and are challenging to study by structural methods (27,28). Therefore, detailed structural information about RGG recognition of G-quadruplexes has been mostly obtained from studies on sc1 RNA (21), the RNA selected against the entire human FMRP but shown to require only the RGG motif of the protein for high affinity and specific binding (29,37,38).…”

“…Some of these RNAs harbor motifs that may form G-quartets and G-quadruplexes in vivo (21, 25). Several mRNAs contain regions shown to form G-quadruplexes implicated in FMRP binding in vitro (21,24,(26)(27)(28)(29). Notably, binding of FMRP to G-rich RNAs in vitro requires only the RGG motif, which specifically interacts with natural and in vitro selected G-quadruplex-containing RNAs such as a 35-nucleotide sc1 RNA (21,(26)(27)(28)(29).…”

“…Several mRNAs contain regions shown to form G-quadruplexes implicated in FMRP binding in vitro (21,24,(26)(27)(28)(29). Notably, binding of FMRP to G-rich RNAs in vitro requires only the RGG motif, which specifically interacts with natural and in vitro selected G-quadruplex-containing RNAs such as a 35-nucleotide sc1 RNA (21,(26)(27)(28)(29). Recent studies showed that G-quadruplexes facilitate mRNA interactions with ribosome-bound FMRP (30), whereas the RGG motif, in addition to mRNA binding, contributes to association with ribosomes and proteins and translational control (14,(31)(32)(33)(34).…”